Method of Obtaining a Hydroxytyrosol-Rich Composition From Vegetation Water

ABSTRACT

The invention provides olive-derived hydroxytyrosol. According to one aspect of the invention, vegetation water is collected from olives. Acid is added to stabilize the vegetation water and prevent fermentation. The mixture is incubated to allow oleoeuropein to convert to hydroxytyrosol, and then fractionated to separate hydroxytyrosol from other components. The hydroxytyrosol is useful as a natural anti-bacterial, anti-viral and fungicidal product for agricultural and pest control applications. In addition, it is useful as a therapeutic and anti-oxidant for a variety of health purposes.

This application claims the benefit of U.S. Provisional Application No.60/230,535 filed Sep. 1, 2000, which is expressly incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

This invention relates to a phenolic fraction of a group of compoundspresent in olive plants known as hydroxytyrosol(3,4-dihydroxyphenylethanol). Particularly, the invention provides anolive extract containing hydroxytyrosol, with low amounts orsubstantially free of oleoeuropein and tyrosol, and a method ofobtaining the same.

REFERENCES

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Braga, C., et al., Cancer 82:448-453 (1998).

Chan, J. M., et al., Seminars in Cancer Biology 8:263-273 (1998).

d'Amicis, A. and Farchi, S., in: Advances in Nutrition and Cancer 2(Zappia, V., et al., Eds.) 67-72, Kluwer Academic/Plenum Publishers, NewYork (1999).

Deiana, M., et al., Free Radic. Biol. Med. 26:762-769 (1999).

de la Puerta, R., et al., Biochem. Pharmacol. 57:445-449 (1999).

Ficarra, P., et al., Farmaco 46:803-815 (1991).

Gerber, M., Epidemiology of Diet and Cancer, ed. M. J. Hill, 263-275(1994).

Kohyama, N., et al., Biosci. Biotechnol. Biochem. 61:347-350 (1997).

Kuller, L. H., Journal of the American Dietetic Association 97:S9-S15(1997).

La Vecchia, C., et al., European Journal of Cancer Prevention 7:461-464(1998).

Manna, C., et al., FEBS Letters 470:341-344 (2000).

Martin-Moreno, J. M., et al., Int. J. Cancer 58:774-780 (1994).

Mattson, F. H. and Grundy, S. M., J. Lipid Res. 26:194-202 (1985).

Owen, R. W., et al., J. Can. Res. Clin. Onc. 125:S31 (2000a).

Owen, R. W., et al., Eur. J. Cancer 36:1235-1247 (2000b).

Owen, R. W., et al., Food Chem. Toxic. 38:647-659 (2000c).

Parthasarathy, S., et al., Proc. Natl. Acad. Sci. USA 87:3894-3898(1990).

Petroni, A., et al., Thromb. Res. 78:151-160 (1995).

Risch, H. A., et al., Journal of the National Cancer Institute86:1409-1415 (1994).

Romani, A., et al., J. Agric. Food Chem. 47:964-967 (1999).

Tsimidou, M., et al., Food Chem. 44:53-60 (1992).

Visioli, F., et al., FEBS Letters 468:159-160 (2000).

Visioli, F. and Galli, C., Nutr. Rev. 56:142-147 (1998).

BACKGROUND OF THE INVENTION

A high amount of dietary fat has been implicated in the development ofseveral diseases (Owen et al., 2000c). Atherosclerosis (Kuller, 1997)and coronary heart disease (Gerber, 1994), as well as cancer of thebreast (La Vecchia et al., 1998), prostate (Chan et al., 1998), ovary(Risch et al., 1994), and colon (Armstrong and Doll, 1975) have eachbeen associated with elevated dietary fat. However, evidence indicatesthat it is not only the amount, but also the type of dietary fat that isimportant in the etiology of some cancers (Bartsch et al., 1999).

Olive oil, the principal fat component of the Mediterranean diet, hasbeen associated with a lower incidence of coronary heart disease (Owenet al., 2000b; Parthasarathy et al., 1990; Mattson and Grundy, 1985) andcertain cancers (d'Amicis and Farchi, 1999; Braga et al., 1998;Martin-Moreno et al., 1994). Several laboratories have reported that thehydrolysis of the olive oil phenolics oleuropin and other family memberslead to small phenolic components with strong chemoprotective activity(Owen et al., 2000a; Manna et al., 2000). In particular, the olive oilphenolic hydroxytyrosol prevents low density lipoprotein (LDL) oxidation(Visioli and Galli, 1998), platelet aggregation (Petroni et al., 1995),and inhibits 5- and 12-lipoxygenases (de la Puerta et al., 1999; Kohyamaet al., 1997). Hydroxytyrosol has also been found to exert an inhibitoryeffect on peroxynitrite dependent DNA base modification and tyrosinenitration (Deiana et al., 1999), and it counteracts cytotoxicity inducedby reactive oxygen species in various human cellular systems (Manna etal., 2000). Finally, studies have shown that hydroxytyrosol isdose-dependently absorbed in humans following ingestion, indicating itsbioavailability (Visioli et al., 2000).

Conventionally, olive oil production involves crushing olives, includingthe pits, to produce a thick paste. During this procedure, the crushedolives are continuously washed with water, a process known as“malaxation.” The paste is then mechanically pressed to squeeze out theoil content. In addition to providing olive oil, the pressing alsosqueezes out the paste's water content. Such washing and pressing stepsyield a considerable amount of water, referred to as “vegetation water.”

Both the pit and the pulp of olives are rich in water-soluble, phenoliccompounds. Such compounds are extracted from olives during malaxation,according to their partition coefficients, and end up in the vegetationwater. This explains why various phenolic compounds, such asoleoeuropein and its derivatives, produced in olive pulp, can be foundin abundance in vegetation waters. Similarly, a number of monophenoliccompounds, such as tyrosol and its derivatives, produced in olive pits,are also abundant in vegetation waters.

Because of the strong chemoprotective activity of hydroxytyrosol, it isdesirable to develop a method which produces an aqueous olive extractwith a high percentage of hydroxytyrosol.

SUMMARY OF THE INVENTION

In one aspect, the invention includes a method of producing ahydroxytyrosol-rich composition. The method has the steps of (a)producing vegetation water from olives, preferably depitted olive meat,(b) adding acid to the vegetation water in an amount effective toproduce a pH between 1 and 5, preferably 2-4, and (c) incubating theacidified vegetation water for a period of at least two months,typically 6-12 months until at least 75%, and preferably at least 90% ofthe oleoeuropein originally present in the vegetation water has beenconverted to hydroxytyrosol.

In one embodiment, the incubating is carried out until the vegetationwater has a weight ratio of hydroxytyrosol to oleoeuropein of between5:1 and 200:1, preferably 10:1 and 100:1. In a related embodiment, theincubating is carried out until the vegetation water has a weight ratioof hydroxytyrosol and tyrosol of between 3:1 and 50:1, typically 5:1 to30:1.

The method may further include fractionating the incubated, vegetationwater to separate hydroxytyrosol from other components, and/or dryingthe vegetation water of isolated hydroxytyrosol to produce a driedextract.

In another aspect, the invention includes a method of producing ahydroxytyrosol-rich composition that includes the steps of (a) producingvegetation water from olives; (b) optionally, drying the vegetationwater; (c) contacting the optionally dried vegetation water with asupercritical fluid; and (d) recovering the hydroxytyrosol-richcomposition from the contacted vegetation water. In one embodiment, thehydroxytyrosol-rich composition includes at least about 95 percent byweight hydroxytyrosol. In another embodiment, the hydroxytyrosol-richcomposition includes at least about 97 percent by weight hydroxytyrosol.In yet another embodiment, the hydroxytyrosol-rich composition includesat least about 99 percent by weight hydroxytyrosol.

In one embodiment, the recovering step described above includes thesteps of (a) recovering the supercritical fluid, where the supercriticalfluid contains the hydroxytyrosol; and (b) vaporizing the supercriticalfluid to extract the hydroxytyrosol-rich composition. In anotherembodiment, the contacting step described above comprises the steps of(a) providing a porous membrane having opposite sides in a module underpressure with the membrane serving as a barrier interface between afluid and a dense gas, the membrane being nonselective for saidhydroxytyrosol; (b) providing the supercritical fluid into the module onone side of the membrane and the vegetation water on the opposite sideof the membrane; (c) and extracting the hydroxytyrosol across themembrane as driven by a concentration gradient of the hydroxytyrosolbetween the vegetation water and the supercritical fluid. In oneembodiment, the porous membrane is a hollow fiber membrane. In anotherembodiment, the supercritical fluid is carbon dioxide.

In another aspect, the invention includes a dietary supplementcomprising an aqueous extract of olives containing a weight ratio ofhydroxytyrosol to oleoeuropein of between 5:1 and 200:1, typically 10:1and 100:1.

In a related aspect the invention includes a dietary supplementcomprising an aqueous extract of olives containing a weight ratio ofhydroxytyrosol and tyrosol of between 3:1 and 50:1, typically 5:1 and30:1.

The above supplements may be dried to provide a powder extract, whichcan formulated into a tablet, capsule, pill, or confection foodadditive.

These and other objects and features of the invention will be more fullyappreciated when the following detailed description of the invention isread in conjunction with the accompanying figure and tables.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 shows the structures of phenolic compounds and their precursorsdetected in olive oil: ligstroside (I); oleuropein glucoside (II);aglycone of ligstroside (III); aglycone of oleuropein glucoside (IV);dialdehydic form of ligstroside aglycone laking a carboxymethyl group(V); dialdehydic form of oleuropein glucoside aglycone lacking acarboxymethyl group (VI); tyrosol (VII); hydroxytyrosol (VIII).

FIG. 2 shows the HPLC analysis of a hydroxytyrosol-rich composition ofthe invention after supercritical carbon dioxide extraction fromvegetation water.

FIG. 3 shows the HPLC analysis of a hydroxytyrosol-rich composition ofthe invention following supercritical carbon dioxide extraction, withsynthetic hydroxytyrosol.

FIG. 4 shows the mass spectrum of a hydroxytyrosol-rich composition ofthe invention.

FIG. 5 illustrates the fragmentation pathway of hydroxytyrosol.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

Unless otherwise indicated, all terms used herein have the same meaningas they would to one skilled in the art of the present invention. It isto be understood that this invention is not limited to the particularmethodology, protocols, and reagents described, as these may vary.

By “oleoeuropein” is intended secoiridoid glucoside oleuropein(Structure II in FIG. 1).

By “tyrosol” is intended 4-hydroxyphenethyl acohol (Structure VII inFIG. 1).

By “hydroxytyrosol” is intended 3,4-dihydroxyphenethyl alcohol(Structure VIII in the FIG. 1).

II. Method of the Invention

The invention provides, in one aspect, provides a hydroxytyrosol-richcomposition from olive-derived vegetation water. It has been discoveredthat under specific conditions, as described below, hydroxytyrosol maybe obtained from the vegetation water of olives. Considered below arethe steps in practicing the invention.

A. Producing Vegetation Water

The method of the invention employs olives that may be obtained fromconventional and commercially available sources such as growers.Preferably, the vegetation water is obtained from pitted olives. Theolives processed according to the method disclosed herein may be pittedby any suitable means. Pits in the olives contain tyrosol which is anundesired component in the vegetation water and which may not beappreciably broken down by the acid treatment described below. The pitsmay be separated from the pulp manually or in an automated manner asdescribed below. Preferably, such means should be capable of segregatingthe pits without breaking them, which might otherwise cause higherconcentrations of tyrosol in the vegetation water. In anotherembodiment, hydroxytyrosol is extracted from vegetation water obtainedfrom olives that have not been pitted.

To produce vegetation water, olive pulp from the olives is first pressedto obtain a liquid-phase mixture including olive oil, vegetation water,and solid by-products. Thereafter, the vegetation water is separatedfrom the rest of the liquid phase mixture and collected. Exemplarymethods of obtaining vegetation water are described in co-owned U.S.Pat. Nos. 6,165,475 and 6,197,308, both to R. Crea, each of which areexpressly incorporated herein by reference in their entirety.

For purposes of commercial production, it may be desirable to automatevarious aspects of the invention. In this regard, one embodimentcontemplates the use of an apparatus as disclosed in U.S. Pat. Nos.4,452,744, 4,522,119 and 4,370,274, each to Finch et al., and eachexpressly incorporated herein by reference. Briefly, Finch et al. teachan apparatus for recovering olive oil from olives. Initially, olives arefed to a pulper that separates the olive pits from the olives to obtaina pitless olive meat. The meat is then taken up by an extraction screwthat subjects the meat to an extraction pressure sufficient to withdrawa liquid phase, comprising oil, water and a minor proportion of olivepulp. The liquid phase is collected in a bin and then sent to aclarifying centrifuge that separates the pulp from the liquid phase toobtain a mixture comprising olive oil and vegetation water. A purifyingcentrifuge then separates the vegetation water and a small proportion ofsolid matter from the mixture to obtain an olive oil, substantially freeof vegetation water, that is collected in a tank. According to Finch etal., the water is put to a disposal means such as a sewer. The presentinvention, in sharp contrast, provides for the collection, saving anduse of the vegetation water to extract hydroxytyrosol.

Additional devices that may be used in practicing the present inventionare disclosed in Italian Patent Nos. 1276576 and 1278025, each of whichis expressly incorporated herein by reference. As above, these devicescan be used to separate the pulp from the pits prior to processing ofthe crushed olive pulp into oil, water, and solid residues.

B. Conversion of Oleoeuropein to Hydroxytyrosol

In one aspect of the invention, the oleoeuropein contained in thevegetation water is converted to hydroxytyrosol. The pH of thevegetation water may be decreased by the addition of acid, and thevegetation water allowed to incubate under conditions which, accordingto the discovery of the invention, promote acid hydrolysis ofoleoeuropein to hydroxytyrosol. The sample may then be fractionated toseparate hydroxytyrosol from other compounds.

In a preferred embodiment, the added acid is citric acid. The acid isadded to the vegetation water to adjust the pH to 1-5, preferably 2-4.Solid citric acid can be added while continuously stirring in an amountof preferably about 25 to 50 pounds of acid per about 1000 gallons ofvegetation water. The pH of the resulting solution can be monitored, andfurther addition of acid may be necessary to achieve the desired pH.Exemplary methods showing the conversion of oleoeuropein tohydroxytyrosol following the addition of citric acid are given inExamples 1 and 2.

The acid may also be an organic or inorganic acid other than citricacid. Exemplary acids which may be used in the present invention includethe inorganic substances known as the mineral acids—sulfuric, nitric,hydrochloric, and phosphoric acids—and the organic compounds belongingto the carboxylic acid, sulfonic acid, and phenol groups. The additionof acid to the vegetation water serves several purposes: (i) itstabilizes the vegetation water; (ii) it prevents fermentation of thevegetation water; and (iii) it slowly hydrolizes the oleouropein,converting it to hydroxytyrosol, as shown in Examples 1 and 2. Tables 1and 2, in Examples 1 and 2, respectively, contain data from two samplesof vegetation water and the respective percent composition of variouscomponents in the samples over time following the addition of citricacid. In one embodiment, the mixture is allowed to incubate untilhydroxytyrosol is 75-90% of the total combination of tyrosol andhydroxytyrosol, and substantially none of the oleoeuropein in theoriginal mixture remains.

C. Purification of Hydroxytyrosol

Following the conversion of oleouropein to hydroxytyrosol, the incubatedvegetation water may be fractionated by a number of methods known in theart. Alternatively, vegetation water may be fractionated prior totreatment with acid. Exemplary methods of fractionation includepartitioning with an organic solvent, high pressure liquidchromatography (HPLC), or supercritical fluids.

Vegetation water obtained as described above provides a solution whichis rich in low molecular weight polyphenols, particularly hydroxytyrosoland a small amount of tyrosol. The concentration of hydroxytyrosol inthe processed water may range from 4-5 grams per liter to 10-15 gramsper liter depending upon the degree of dilution during the olive oilextraction. In one embodiment, the invention provides a method ofextraction or purification that selectively enriches the content ofhydroxytyrosol without the addition of contaminants. Thus, the majorpolyphenolic component, hydroxytyrosol, is isolated from other membersof the polyphenolic family, impurities, suspended solids, tannins, andother molecules contained in the vegetation water. Hydroxytyrosol maytherefore be produced in a purity and quantity not readily available bycurrent synthetic or natural extraction methods.

A supercritical fluid is a gas that becomes very dense above itscritical temperature and pressure. Its properties are between those of agas and liquid, resulting in increased ability to dissolve compounds.Its relatively high density, high diffusivity, and low viscosity allowit to extract compounds faster than conventional liquid solvents. Carbondioxide is the gas used most widely for supercritical fluid processingof foods and food ingredients because it is natural, nontoxic,non-flammable, and relatively inert and leaves no residue in theextracted product. Typical liquid extraction with supercritical carbondioxide is usually done by dispersing one phase in the other in largecontacting columns or towers, where the solute containing fluid, such asjuices, flows downward by gravity, and the supercritical carbon dioxideflows upward. Mass transfer occurs at the interface between the twophases.

Alternatively, continuous extraction of liquids and suspensions can beachieved using supercritical fluids, such as carbon dioxide, and porousmembranes instead of contacting columns. Instead of dispersing thephases, the liquid is fed continuously through porous polypropylenemembranes configured as hollow fiber bundles or spiral wound sheets. Theliquid passes through the porous membranes within a pressurized module,while supercritical carbon dioxide flows countercurrently on the otherside of the membrane. The pressure in the module is essentially thesame, so that the extraction is driven by the concentration gradientbetween the fluid and the supercritical carbon dioxide. The extract maybe recovered by vaporizing the carbon dioxide for recycling. Anexemplary method of extraction using supercritical carbon dioxide andporous membranes is described in U.S. Pat. No. 5,490,884, which isexpressly incorporated by reference herein in its entirety.

Other supercritical fluids, instead of, or in combination with, carbondioxide. These fluids include methane, ethane, propane, butane,isobutane, ethene, propene, hydrofluorocarbons, tetrafluoromethane,chlorodifluoromethane, carbon dioxide, dinitrogen monoxide, sulphurhexafluoride, ammonia, and methyl chloride.

Example 3 describes a small scale experiment in support of theinvention, wherein hydroxytyrosol was isolated from vegetation waterusing supercritical carbon dioxide and porous membranes. HPLC and massspectrometry analysis of the isolated hydroxytyrosol shows the sample tobe 97-99% pure hydroxytyrosol. Thus, the invention provides ahydroxytyrosol-rich composition containing at least about 80%hydroxytyrosol, preferably at least about 90% hydroxytyrosol, morepreferably at least about 95% hydroxytyrosol, even more preferably atleast about 97% hydroxytyrosol, and yet, even more preferably at leastabout 99% hydroxytyrosol.

Prior to extraction with a supercritical fluid the vegetation water mayhave carriers, which are known to those of skill in the art, such asmaltodextran and/or polypropylene beads, added to the solution; and/orthe solution may be dried. The drying step preferably removes at leastabout 90%, more preferably at least about 95%, and even more preferablyat least about 98% of the water from the vegetation water.

An important feature of membrane reactors is the fact that contactsurface interfacial area can be added independently of fluid velocities.Accordingly, the invention contemplates a large scale unit where thesurface membrane area of the membrane used for extraction is at leastabout 100 square yards, preferably at least about 300 square yards, andeven more preferably at least about 600 square yards to allow separationof hydroxytyrosol from large volumes of vegetation water. Thus, themembrane system of the invention would, in one aspect, be able toaccommodate a flow rate of between 1-20 liters per minute, preferablybetween 5-10 liters per minute.

Additional purification methods may also be used in accordance with theinvention as mentioned above. HPLC isolation of hydroxytyrosine isdescribed in: Ficarra et al., 1991; Romani et al., 1999; and Tsimidou,1992, each of which is expressly incorporated by reference herein.

III. Hydroxytyrosol-Rich Dietary Supplement

It should be appreciated that hydroxytyrosol produced by the methoddescribed above may be used for a variety of applications. For example,hydroxytyrosol obtained by the method of the present invention can beused: (i) as a natural anti-bacterial, anti-viral and/or fungicidalproduct for agricultural and/or pest control applications, and (ii) as atherapeutic and/or an anti-oxidant for a variety of health purposes. Inone exemplary embodiment, the hydroxytyrosol, is administered to amammalian subject, such as a person desirous of one or more of thebenefits associated with hydroxytyrosol.

The hydroxytyrosol obtained by the method of the invention can beadministered orally or parenterally. Oral dosage forms can be in a solidor liquid form. Such dosage forms can be formulated from purifiedhydroxytyrosol or they can be formulated from aqueous oraqueous-alcoholic extracts. Regarding the latter, aqueous oraqueous-alcoholic (e.g., water-methanol or water-ethanol) extracts canbe spray-dried to provide a dry powder that can be formulated into oraldosage forms with other pharmaceutically acceptable carriers. Theaqueous or aqueous-alcoholic extracts can be formulated to containvarious weight ratios of hydroxytyrosol to oleoeuropein of between 5:1and 200:1, preferably between about 10:1 and about 100:1. The extractsmay also be formulated to contain various weight ratios of hydroxytysoland tyrosol of between about 3:1 and about 50:1, preferably betweenabout 5:1 and about 30:1.

The solid oral dosage form compositions in accordance with thisinvention are prepared in a manner well known in the pharmaceuticalarts, and comprise hydroxytyrosol in combination with at least onepharmaceutically acceptable carrier. In making such compositions, ahydroxytyrosol-rich composition, either in substantially pure form or asa component of a raw distillate or extract, is usually mixed, diluted orenclosed with a carrier. The carrier can be in a solid form, semi-solidor liquid material which acts as a vehicle, carrier or medium for theactive ingredient. Alternatively, the carrier can be in the form of acapsule or other container to facilitate oral administration. Thus, thesolid oral dosage forms for administration in accordance with thepresent invention can be in the form of tablets, pills, powders or softor hard gelatin capsules.

Alternatively, the hydroxytyrosol obtained in accordance with thisinvention for oral administration can be in liquid form wherein thepharmaceutically acceptable carrier is water or an aqueous-alcoholicmedium.

The compositions for administration in the present invention can also beformulated with other common pharmaceutically acceptable excipients,including lactose, dextrose, sucrose, sorbitol, mannitol, starches,gums, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, methylcellulose, water, alcohol and the like. Theformulations can additionally include lubricating agents such as talc,magnesium stearate and mineral oil, wetting agents, emulsifying andsuspending agents, preserving agents such as methyl- andpropylhydroxybenzoates, sweetening agents or flavoring agents. Further,the compositions of the present invention can be formulated so as toprovide quick, sustained or delayed release of the active ingredientafter administration to a subject.

Parenteral formulations for use in accordance with the present inventionare prepared using standard techniques in the art. They are commonlyprepared as sterile injectable solutions, using a parenterallyacceptable carrier such as isotonic saline solution or as a sterilepackaged powder prepared for reconstitution with sterile buffer orisotonic saline prior to administration to a subject.

From the foregoing, it can be seen how various objects and features ofthe invention are met. Those skilled in the art can now appreciate fromthe foregoing description that the broad teachings of the presentinvention can be implemented in a variety of forms. Therefore, whilethis invention has been described in connection with particularembodiments and examples thereof, the true scope of the invention shouldnot be so limited. Various changes and modification may be made withoutdeparting from the scope of the invention, as defined by the appendedclaims.

The following examples illustrate methods of producinghydroxytyrosol-rich compositions in accordance with the invention. Theexamples are intended to illustrate, but in no way limit, the scope ofthe invention.

EXAMPLES Example 1 Conversion from Oleoeuropein to HydroxytyrosolFollowing the Addition of About 25 Pounds of Citric Acid/1000 Gallons

Table 1 shows the conversion of oleoeuropein to hydroxytyrosol over timefollowing the addition of about 25 pounds of citric acid per 1000gallons of vegetation water. The percentages in Table 1 are shown asweight percentages of the total phenolic compounds in the solution. Asdemonstrated in Table 1, hydroxytyrosol comprises over 80% of thephenolic compounds in the solution after 12 months. TABLE 1 Conversionfrom Oleoeuropein to Hydroxytyrosol Following the Addition of About 25Pounds of Citric Acid/1000 Gallons Compo- Compo- Compo- Compo- sition atsition at sition at sition at Component T = 2 mo. T = 3 mo. T = 4.5 mo.T = 12 mo. Hydroxytyrosol 30.4%  32% 48.4%  80.2%  Tyrosol 2.5%  5% 2.2%3.6% Oleoeuropein  41% 36.6%  25.1%  1.2% Oleoeuropein 4.2% 4.6%  2.7%3.7% aglycone

Example 2 Conversion from Oleoeuropein to Hydroxytyrosol Following theAddition of About 50 Pounds of Acid/1000 Gallons

Table 2 shows the conversion of oleoeuropein to hydroxytyrosol over timefollowing the addition of about 50 pounds of citric acid per 1000gallons of vegetation water. The percentages in Table 2 are shown asweight percentages of the total phenolic compounds in the solution.Significantly, as demonstrated in Table 2, hydroxytyrosol comprises over45% of the phenolic compounds in the solution after 2 months. TABLE 2Conversion from Oleoeuropein to Hydroxytyrosol Following the Addition ofAbout 50 Pounds of Acid/1000 Gallons Composition Composition Componentat T = 2 mo. at T = 12 mo. Hydroxytyrosol 45.7%  78.5%  Tyrosol 2.9%3.3% Oleoeuropein 28.7%  1.5% Oleoeuropein aglycone 4.1% 3.5%

Example 3 Extraction of Hydroxytyrosol from Vegetation Water

An aliquot (0.5 ml) of vegetation water containing about 40 mg of drysolid (maltodextran) was mixed with polypropylene porous beads anddried. The dry mix was used for extraction with supercritical carbondioxide (PoroCrit, LLC, Berkeley, Calif.). The collected sample (about2.0 mg) was analyzed by HPLC. The profile of the sample is shown in FIG.2, and Table 3 shows the area under the major peak to be 97%. Whensynthetic hydroxytyrosol was added to the sample and analyzed by HPLC,one major peak appeared, as shown in FIG. 3, indicating that the majorproduct of the sample is hydroxytyrosol (Table 4).

Mass spectrometry analysis of the sample, as shown in FIG. 4, confirmedthat the major product is hydroxytyrosol. The sample was diluted to afinal concentration of 26 micrograms per milliliter with methanol andanalyzed in negative ionization mode on a Finnigan LCQ fitted with anESI probe. The infusion was at 3 microliters per minute using anintegrated syringe pump. The temperature was 270 C, needle voltage +4.2V, sheath gas 45 units, and auxiliary gas 10 units. The fragmentationpathway of hydroxytyrosol is shown in FIG. 5. As can be seen in FIG. 4,hydroxytyrosol (mass/charge 153.1) and its fragmentation products (123.1and 105.1 mass/charge) account for the majority of the product abundancein the multi-stage spectrum. TABLE 3 Peak Analysis of FIG. 2 HPLCResults Peak No. Time Height (μV) Area (μV-sec) Area (%) 1 5.935 2155426687705 97.476 2 11.433 5686 173104 2.523

TABLE 4 Peak Analysis of FIG. 3 HPLC Results Peak No. Time Height (μV)Area (μV-sec) Area (%) 1 2.875 1345 13895 0.26 2 3.278 1076 14140 0.2653 6.641 211204 5241105 98.240 4 11.961 2587 65811 1.233

1. A method of producing a hydroxytyrosol-rich composition, comprising(a) producing vegetation water from olives; (b) adding acid to thevegetation water in an amount effective to produce a pH between about 1and about
 5. (c) incubating the acidified vegetation water for a periodof at least two months, until at least 75% of oleoeuropein originallypresent in the vegetation water has been converted to hydroxytyrosol. 2.The method of claim 1, wherein said incubating is carried out for aperiod of at least 9 months, and until at least 90% of oleoeuropeinoriginally present in the vegetation water has been converted tohydroxytyrosol.
 3. The method of claim 1, wherein the vegetation wateris produced from depitted olive meat.
 4. The method of claim 3, whereinthe incubating is carried out until the vegetation water has a weightratio of hydroxytyrosol to oleoeuropein of between about 5:1 and about200:1.
 5. The method of claim 4, wherein the incubating is carried outuntil the vegetation water has a weight ratio of hydroxytyrosol tooleoeuropein of between about 10:1 and about 100:1.
 6. The method ofclaim 3, wherein the incubating is carried out until the vegetationwater has a weight ratio of hydroxytyrosol and tyrosol of between about3:1 and about 50:1.
 7. The method of claim 6, wherein the incubating iscarried out until the vegetation water has a weight ratio ofhydroxytyrosol and tyrosol of between about 5:1 to about 30:1.
 8. Themethod of claim 1, which further comprises fractionating the incubatedvegetation water to separate hydroxytyrosol from other components. 9.The method of claim 1, wherein said acid is added in an amount effectiveto produce a pH between about 2 and about
 4. 10. A method of producing ahydroxytyrosol-rich composition, comprising (a) producing vegetationwater from olives; (b) optionally, drying said vegetation water; (c)contacting said optionally dried vegetation water with a supercriticalfluid; and (d) recovering said hydroxytyrosol-rich composition from thecontacted vegetation water, said hydroxytyrosol-rich compositioncomprising at least about 95 percent by weight hydroxytyrosol.
 11. Themethod of claim 10, wherein said recovering comprises the steps of: (a)recovering said supercritical fluid, where said supercritical fluidcontains said hydroxytyrosol; and (b) vaporizing said supercriticalfluid to extract said hydroxytyrosol-rich composition.
 12. The method ofclaim 10, wherein said contacting comprises the steps of: (a) providinga porous membrane having opposite sides in a module under pressure withsaid membrane serving as a barrier interface between a fluid and a densegas, said membrane being nonselective for said hydroxytyrosol; (b)providing said supercritical fluid into said module on one side of saidmembrane and said vegetation water on the opposite side of saidmembrane; (c) extracting said hydroxytyrosol across said membrane asdriven by a concentration gradient of said hydroxytyrosol between saidvegetation water and said supercritical fluid.
 13. The method of claim12, wherein said porous membrane is a hollow fiber membrane.
 14. Themethod of claim 10, wherein said supercritical fluid is carbon dioxide.15. The method of claim 10, wherein said hydroxytyrosol-rich compositioncomprises at least about 97 percent by weight hydroxytyrosol.
 16. Themethod of claim 10, wherein said hydroxytyrosol-rich compositioncomprises at least about 99 percent by weight hydroxytyrosol.
 17. Adietary supplement comprising an aqueous extract of olives containing aweight ratio of hydroxytyrosol to oleoeuropein of between about 5:1 andabout 200:1.
 18. The supplement of claim 17, which has a weight ratio ofhydroxytyrosol to oleoeuropein of between about 10:1 and about 100:1.19. A dietary supplement comprising an aqueous extract of olivescontaining a weight ratio of hydroxytyrosol and tyrosol of between about3:1 and about 50:1.
 20. The dietary supplement of claim 19, containing aweight ratio of hydroxytyrosol and tyrosol of between about 5:1 andabout 30:1.
 21. The dietary supplement of claim 17, wherein saidsupplement dried to provide a powder extract.
 22. The dietary supplementof claim 17, wherein said extract is in the form of a tablet, capsule,pill, or confection food additive.